62 research outputs found
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The Fusarium graminearum Histone H3 K27 Methyltransferase KMT6 Regulates Development and Expression of Secondary Metabolite Gene Clusters
The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the “cryptic genome”, specifically secondary metabolite expression
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Histone modifications rather than the novel regional centromeres of Zymoseptoria tritici distinguish core and accessory chromosomes
Background: Supernumerary chromosomes have been found in many organisms. In fungi, these “accessory” or “dispensable” chromosomes are present at different frequencies in populations and are usually characterized by higher repetitive DNA content and lower gene density when compared to the core chromosomes. In the reference strain of the wheat pathogen, Zymoseptoria tritici, eight discrete accessory chromosomes have been found. So far, no functional role has been assigned to these chromosomes; however, they have existed as separate entities in the karyotypes of Zymoseptoria species over evolutionary time. In this study, we addressed what—if anything—distinguishes the chromatin of accessory chromosomes from core chromosomes. We used chromatin immunoprecipitation combined with high-throughput sequencing (“ChIP-seq”) of DNA associated with the centromere-specific histone H3, CENP-A (CenH3), to identify centromeric DNA, and ChIP-seq with antibodies against dimethylated H3K4, trimethylated H3K9 and trimethylated H3K27 to determine the relative distribution and proportion of euchromatin, obligate and facultative heterochromatin, respectively. Results: Centromeres of the eight accessory chromosomes have the same sequence composition and structure as centromeres of the 13 core chromosomes and they are of similar length. Unlike those of most other fungi, Z. tritici centromeres are not composed entirely of repetitive DNA; some centromeres contain only unique DNA sequences, and bona fide expressed genes are located in regions enriched with CenH3. By fluorescence microscopy, we showed that centromeres of Z. tritici do not cluster into a single chromocenter during interphase. We found dramatically higher enrichment of H3K9me3 and H3K27me3 on the accessory chromosomes, consistent with the twofold higher proportion of repetitive DNA and poorly transcribed genes. In contrast, no single histone modification tested here correlated with the distribution of centromeric nucleosomes. Conclusions: All centromeres are similar in length and composed of a mixture of unique and repeat DNA, and most contain actively transcribed genes. Centromeres, subtelomeric regions or telomere repeat length cannot account for the differences in transfer fidelity between core and accessory chromosomes, but accessory chromosomes are greatly enriched in nucleosomes with H3K27 trimethylation. Genes on accessory chromosomes appear to be silenced by trimethylation of H3K9 and H3K27.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by BioMed Central. The published article can be found at: http://www.epigeneticsandchromatin.com/. Supporting information available online at: http://www.epigeneticsandchromatin.com/content/8/1/41Keywords: Histone methylation, ChIP-seq, Zymoseptoria tritici (Mycosphaerella graminicola), Centromere, Accessory chromosome
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Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans
Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions
Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen
It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits
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ConnollyLanelleBiochemistryBiophysicsFusariumGraminearumHistone_SupportingInformation.zip
The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated
transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput
DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for
trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was
found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or
trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found
in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27
methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by
western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments
and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for
H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set
of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no
enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for
expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence
of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly
involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity
factors. Results from this study provide the framework for novel targeted strategies to control the ‘‘cryptic genome’’,
specifically secondary metabolite expression
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ConnollyLanelleBiochemistryBiophysicsFusariumGraminearumHistone.pdf
The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated
transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput
DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for
trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was
found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or
trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found
in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27
methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by
western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments
and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for
H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set
of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no
enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for
expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence
of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly
involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity
factors. Results from this study provide the framework for novel targeted strategies to control the ‘‘cryptic genome’’,
specifically secondary metabolite expression
The <i>Fusarium graminearum</i> Histone H3 K27 Methyltransferase KMT6 Regulates Development and Expression of Secondary Metabolite Gene Clusters
<div><p>The cereal pathogen <i>Fusarium graminearum</i> produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other <i>Fusarium</i> species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of <i>Drosophila</i> Enhancer of zeste, E(z). The <i>kmt6</i> mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in <i>kmt6</i>. A largely overlapping set of genes showed increased expression in <i>kmt6</i>. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in <i>kmt6</i>. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the “cryptic genome”, specifically secondary metabolite expression.</p></div
Distribution of histone modifications across the complete set of <i>F. graminearum</i> genes.
<p>The left panel for each modification shows read counts (reads/gene) across all genes that were aligned to make the known or presumed transcriptional start site (TSS) position “0”. The middle panel shows read counts across all genes (reads/gene) normalized by gene length (x-axis indicates percent of gene length). The right panel shows the distribution of normalized read counts (NLCS; density) per gene. For most modifications two peaks are observed, background levels of reads (“B”) and enrichment (“E”). Results are for low nitrogen experiments, but indistinguishable results were obtained with samples grown on high nitrogen medium.</p
The <i>kmt6</i> mutant displays altered growth and development.
<p>A. WT and <i>kmt6</i> strains were grown on minimal (MIN) or YPD medium. The mutant shows sparse growth on MIN and dense, slow-growing and intensely orange mycelium on YPD. The lower panel shows YPD plates with a complemented strain (<i>kmt6</i>+<i>kmt6<sup>+</sup></i>) and a plasmid control (<i>kmt6</i>+plasmid). B. WT and <i>kmt6</i> were grown through race tubes on MIN or YPD to measure linear growth for 25 to 33 days. Results are the average of three replicates, and bars indicate standard error. C. Sexual development was induced on carrot agar (CAR) plates. Black patches in the WT plate indicate numerous fully developed perithecia, while no perithecia formed in <i>kmt6</i> plates. The complemented strain (<i>kmt6</i>+<i>kmt6<sup>+</sup></i>) is able to produce perithecia and ascospores at similar levels as WT. Protoplast fusions to complement <i>kmt6 neo<sup>+</sup></i> strains with WT <i>hph<sup>+</sup></i> nuclei do not restore formation of perithecia. These heterokaryons did not break down, as colonies resistant to both G418 and Hyg were isolated from different areas of the CAR plate and grew on YPD+G418+Hyg (center and edge).</p
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